Download C. elegans neuronal regeneration is influenced by life stage, ephrin signaling, and synaptic branching

Caenorhabditis elegans neuronal regeneration
is influenced by life stage, ephrin signaling,
and synaptic branching
Zilu Wu*†‡, Anindya Ghosh-Roy*, Mehmet Fatih Yanik§, Jin Z. Zhang¶, Yishi Jin*†‡, and Andrew D. Chisholm*†储
*Division of Biological Sciences, Center for Molecular Genetics, and ‡Howard Hughes Medical Institute, University of California at San Diego,
La Jolla, CA 92093; †Department of Molecular, Cell, and Developmental Biology, Sinsheimer Laboratories, University of California,
Santa Cruz, CA 95064; §Department of Physics, Stanford University, Palo Alto, CA 94305; and ¶Department of Chemistry
and Biochemistry, University of California, Santa Cruz, CA 95064
Communicated by Cornelia I. Bargmann, The Rockefeller University, New York, NY, July 26, 2007 (received for review May 15, 2007)
We previously reported functional regeneration of Caenorhabditis
elegans motor neurons after femtosecond laser axotomy. We
report here that multiple neuronal types can regrow after laser
axotomy using a variety of lasers. The precise pattern of regrowth
varies with cell type, stage of animal, and position of axotomy.
Mechanosensory axons cut in late larval or adult stages displayed
extensive regrowth, yet failed to reach their target area because of
guidance errors in the anteroposterior axis. By contrast, mechanosensory axons cut in early larval stages regrew at the same rate
but with fewer anteroposterior guidance errors, and were more
likely to reach their target area. In adult animals lacking the VAB-1
Eph receptor tyrosine kinase, mechanosensory axon regrowth was
more accurate than in the wild type, suggesting that guidance
errors of regrowing touch neuron axons are the result of Eph
signaling. Kinase-dependent and kinase-independent Eph signaling influenced outgrowth and guidance of regrowing touch neurons, respectively. Mechanosensory neurons regrew when severed
proximal to their collateral synaptic branch but did not regrow
when severed distal to the branch point. However, the distal axon
could regrow if the branch is removed surgically at the same time
as distal axotomy, or at a later time. The touch neuron synaptic
branch point may act as a sorting area to regulate growth. These
findings reveal that multiple influences affect regenerative growth
in C. elegans neurons.
We are interested in the fundamental conserved aspects of
neuronal regeneration. Until recently, there has been little
analysis of axon regeneration in genetically tractable model
organisms. Although myelinated, Zebrafish CNS axons display
spontaneous regeneration (13), studies of which have revealed
the importance of cAMP in promoting regrowth in vivo (14).
Needles have been used to generate large-scale brain injuries to
study the responses of Drosophila central neurons to damage
(15), and surgical removal of sense organs has been used to
analyze degenerative processes in sensory neurons (16).
We previously used an amplified femtosecond laser to cut
identified GFP-labeled axons in Caenorhabditis elegans and
found that motor neurons can regrow and restore function (17).
Femtosecond laser surgery has also been used to cut the sensory
dendrites of C. elegans chemosensory neurons although these
were found not to regenerate (18). Here, we report that multiple
neuron types in C. elegans regrow in response to axotomy, using
a variety of laser types. However, C. elegans neurons are not fully
competent for regeneration. We report that regrowth responses
are strongly influenced by larval stage. Eph signaling influences
the accuracy of regeneration in adult mechanosensory neurons.
Finally, we show that the synaptic branch point of mechanosensory neurons defines a transition point in the regrowth potential
of an axon.
Results
axotomy 兩 laser 兩 femtosecond laser 兩 microsurgery
R
egeneration of neuronal processes after injury has been
studied at the cellular level since the days of Ramon y Cajal
(1). Axons of most vertebrates and invertebrates display strong
regenerative responses. Axons that successfully regenerate can
form a new growth cone at the cut tip of the axon within hours
of damage. Formation of a new growth cone after injury involves
elevation of intracellular calcium (2), several intracellular signaling pathways (3), and local protein synthesis (4). Axotomyinduced signals may feed into growth cone initiating pathways
related to those used in developmental collateral branching. The
regenerated growth cone then undergoes a transition from
sprouting to elongation growth mode; regenerating and developmental axon growth may involve both common and distinct
molecular pathways (5).
Central neurons in mammals and birds fail to regenerate after
axotomy, partly because of the inhibitory environment of the
CNS (6). Intensive analysis has identified several inhibitory
components of myelin (7), as well as of the astrocytic glial scar
(8). The inhibitory components of mammalian myelin interact
with a neuronal receptor complex (9, 10) that represses axon
growth via the small GTPase Rho (11). Inhibitory influences of
the CNS environment are not absolute, and can be overcome by
various treatments, such as conditioning peripheral lesions that
increase the intrinsic ability of CNS axons to regrow (12).
15132–15137 兩 PNAS 兩 September 18, 2007 兩 vol. 104 兩 no. 38
Cutting C. elegans Axons Using Femtosecond and Conventional Lasers.
We previously reported cutting individual neuronal processes in
C. elegans using an amplified femtosecond laser (17). We have
since found that axons can be cut using a high repetition rate
unamplified femtosecond laser (80–90 MHz) mode or a conventional UV laser of the type used for C. elegans cell ablations
(19). There are several differences in the immediate responses of
axons cut with the different lasers [supporting information (SI)
Table 1 and SI Fig. 5]. High repetition rate (‘‘MHz’’) femtosecond laser axotomy creates a gap of 2–5 ␮m within seconds of
cutting, whereas low repetition rate (‘‘kHz’’) laser axotomy is
more precise, creating smaller gaps (1–2 ␮m; see SI Movie 1).
These gaps expand over several hours because of retraction of
both cut ends. Unamplified MHz or UV lasers often create
damage to the surrounding epidermis visible as autofluorescent
Author contributions: Y.J. and A.D.C. designed research; Z.W. and A.G.-R. performed
research; Z.W., A.G.-R., M.F.Y., and J.Z.Z. contributed new reagents/analytic tools; Z.W.,
A.G.-R., and A.D.C. analyzed data; and Y.J. and A.D.C. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Abbreviations: AP, anteroposterior; CD, cavity dumped; FWHM, full width half maximum.
储To
whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/
0707001104/DC1.
© 2007 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0707001104
commissure X
DD1 + VD2
X
VD3
DD2
VD4
X
ventral process DD
ventral cord
VD
L4 + 8 h
B
DD4
X
df
+ 30 h
+ 30 h
*
*
DD4
vulva
C
DD5
DD5
L3 +10 h
*
+ 24 h
*
*
DD4
D
df
DD5
DD4
L4 0 h
DD5
+9h
df
df
DD5
*
*
+ 39 h
*
Fig. 1. Regrowth of D motor neurons after axotomy. (A) D neuron morphology and synaptic polarity; triangles indicate neuromuscular junctions; X marks
indicate sites of axotomy in mid lateral commissure and in ventral cord.
Confocal of D neuron morphology in L4 stage, juIs76 marker. (Scale bar: 20
␮m.) (B) When D neuron commissures were cut at the mid lateral position
(arrow), they retracted as far as the lateral edges of the muscle quadrants.
Proximal stumps of cut D commissures became swollen and began to sprout
within 6 –12 h of cutting. Most DD neurons cut in the L4 stage regrew to the
dorsal cord within 8 –10 h (e.g., 21/23 DD5s). In all figures, red asterisks indicate
a regrown process, and df denotes the distal fragment of the cut axon.
Midbody D neurons become unable to regrow dorsally as the reproductive
system develops. Neurons DD4 ⫹ DD5 (marker juIs145) were cut in L4 stage. At
30 h, DD4 has not grown, whereas DD5 has reached the dorsal cord. (C) DD4
and DD5 (marker ynIs37) cut in L3 stage; both regrow to dorsal cord by 10 h.
Note splitting and lateral extension of both regrowing processes as they meet
dorsal muscle (*), similar to the behavior of VD growth cones in development
(31); one of the DD4 branches has been pruned by 24 h. (D) Growth of new DD5
commissure to dorsal cord after cutting in ventral process (arrow, 0 h), L4 stage
juIs145.
scars (SI Fig. 5 A and F), around which the regrowing axon often
extends (e.g., Fig. 3A). After kHz axotomy no scarring is seen;
axons cut in this way occasionally (⬍10%) repair themselves by
fusion with the distal fragment within 6 h of cutting. By using kHz
lasers, axons can be cut with only 10 pulses of 20 nJ each. Axons
cut using any of the three laser types regrow after a lag period
of 6–24 h depending on the cell type; the rate of regrowth and
the total distance grown after injury were not significantly
different between axons cut with the different lasers (SI Fig. 5E).
In the analyses presented here we chose to use femtosecond
lasers in the MHz mode.
Motor Neurons and Chemosensory Neurons Can Regrow After Axotomy. In our earlier studies, we reported that GABAergic D type
motor neurons (Fig. 1A) showed robust regeneration when
severed in the L4 stage using an amplified femtosecond laser
(17). We confirmed that ⬇70% of DD and VD commissures cut
in the L4 stage regrew after axotomy using the MHz femtosecond laser (Fig. 1B; n ⫽ 49). Axotomized D neuron commissures
formed swollen tips or growth cones by 8–12 h and thereafter
extended dorsally at rates of 2–3 ␮m/h. We found no consistent
differences in the ability of DD and VD neurons to regrow, with
the exception of the midbody neurons DD4 and VD8. DD4 and
VD8 either did not extend sprouts or grew laterally and ventrally
after cutting; regrowing DD4 or VD8 cells rarely reached the
dorsal midline (2/23 DD4 and 1/9 VD8 reached the dorsal cord
Wu et al.
at 24 h; Fig. 1B). Two cells that develop in the midbody of the
L4 hermaphrodite, the uterine seam cell and the vul E cell, are
connected to the lateral epidermis (20) and might physically
block the regrowth of nearby commissures.
To test whether the development of these structures might
account for the inability of DD4/VD8 to regrow, we cut DD cells
in the L2 or L3 stages and found that all DD cells, including DD4,
could regrow to the dorsal midline (5/6 DD4s regrew by 10 h; Fig.
1C). The failure of midbody motor commissures to regenerate in
the L4 stage is likely a result of the development of the
reproductive structures.
In these experiments, commissures were cut in mid lateral
positions, 20–30 ␮m from their target areas in the dorsal cord.
To test whether motor neurons could regenerate when cut
further from their target area we severed DD axons within the
ventral nerve cord of L4 animals, between the cell body and the
commissural branch (Fig. 1 A). Three of six DD5s extended new
commissures to the dorsal midline (Fig. 1D), showing that DD
neurons can regrow an entire commissure after injury.
To explore the generality of these findings we next tested the
regenerative responses of chemosensory neurons, which are
bipolar neurons with ciliated sensory dendrites. Sensory dendrites of dye-filled phasmid neurons do not regrow when cut with
amplified lasers (17, 18). Using unamplified MHz laser cutting
of GFP-marked neurons we confirmed that phasmid processes
did not regrow when cut (SI Fig. 6B). The dendrites of the
ciliated sensory neurons ADF and ASH do not regrow after
amplified femtosecond laser surgery (18). We tested whether
these findings extended to a chemosensory neuron with a
complex cilium, AWB. AWB dendritic processes severed in the
L4 stage frequently regrew (5/11 AWB cells showed sprouting),
although more slowly than motor or mechanosensory neurons
(SI Fig. 6 D and E). Regrowing AWB processes extended
anteriorly and in one case regrew to the tip of the nose. These
observations show that some chemosensory dendrites can regrow after injury.
Accuracy of Mechanosensory Neuron Process Regrowth Declines with
Age. We previously showed that mechanosensory axons can
regrow when cut using amplified femtosecond lasers (21). We
confirmed and extended these observations using MHz axotomy.
Mechanosensory neurons are bipolar, with long anterior sensory
axons and short posterior axons; their synaptic output is from
collateral branches formed from their anterior axons (Fig. 2A).
We first cut ALM axons at ⬃30% of their length (30–50 ␮m from
the ALM cell body); we cut PLMs at similar distances from their
cell bodies. The sequence of changes after axotomy in touch
neurons paralleled that observed in motor neurons: after several
hours the proximal stump swelled and developed a growth cone
with filopodial protrusions between 10 and 24 h. Between 24 and
48 h ALM axons extended at 7.0 ⫾ 0.9 ␮m/h (Fig. 2B). Most
(38/48) ALMs cut in the L4 or in young adults sprouted and
regrew from the proximal stump; in only two cases the axon
regrew from a new collateral branch. Newly formed axons
initially grew anteriorly but randomly strayed dorsally or ventrally; many reversed direction and extended posteriorly (11/18
L4s/young adults; Fig. 2B), and some of these reversed again to
form loops (cf. PLM, Fig. 3A). In some cases the axotomized
axon did not regrow; instead, the uninjured posterior axon grew
out (4/18 L4 ALMs; SI Fig. 7). Regrowing touch neurons
frequently extended additional branches by 24 h, most of which
were pruned by 48 h. For example, 19 PLMs cut in the L4 stage
extended 31 collateral branches at 24 h, 17 of which had
disappeared by 48 h (we define a branch as an extension ⬎2 ␮m).
Nevertheless, although ALM axons regrew 159 ⫾ 23 ␮m in 48 h
after axotomy, they failed to reach their targets in the nerve ring
when cut in L4 or young adult (0/13 L4s; 0/32 young adults).
The response of mechanosensory neurons to axotomy in early
PNAS 兩 September 18, 2007 兩 vol. 104 兩 no. 38 兩 15133
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dorsal cord
A
A
vab-1 zdIs5
Pmec-4-GFP zdIs5
AVM
B
ALML
ALM
zdIs5
ALMR
nr
B
9h
A
PLMR
L1 + 12 h
C
df
L4 0 h
df
25 h
*
ALMR
PLM
ALML + 21 h
56 h
nr
*
+10 h
ALMR
AVM
+ 29 h
df
+ 24 h
*
*
*
+ 48 h
E
ALMR
D
AVM
*
D
ALML
nr
AVM
Fig. 2. Decline in guidance of touch neuron regrowth during larval development. (A) Anatomy of anterior touch neurons ALM and AVM. X, site of
axotomy in ALM, proximal to the synaptic branch in the nerve ring (nr). (B)
Aberrant regrowth of ALML after axotomy in mid L4 stage. A growth cone is
visible at 24 h; by 48 h, the regrown process has reversed posteriorly; the distal
fragment (df) is beaded yet clearly visible at 48 h. (C) Regrowth of ALML after
axotomy in L1 stage. At 12 h, the proximal stump of ALMR has formed a
growth cone but has not yet begun to extend; the distal fragment is faint and
beaded. By 21 h, ALMR has extended anteriorly and ventrally, sending two
branches to the ventral cord (marked by AVM process); the ALMR distal
fragment is no longer visible. By 29 h, ALMR has reached the nerve ring; one
of the ventral cord branches has retracted. (Scale bars: 10 ␮m.) (D) ALML
regrowth into the nerve ring (nr) 24 h after axotomy in L1. The regenerated
ALM (*) lacks the anterior process distal to the synaptic branch. (E) Growth
rates of ALM between 10 and 24 h and 24 and 48 h (gray bars) after axotomy
in different stages; mean ⫾ SEM; n ⬎ 5 for each; only adult growth rates are
significantly slower (t test). *, P ⬍ 0.05.
larval stages was dramatically different from that in later stages.
After axotomy in the L1 stage, 50% (8/16) of ALM axons regrew
to the nerve ring within 24 h. Regrowing axons did not simply
fuse with distal fragments as most took different routes to the
nerve ring, and failed to regrow the anterior extension to the
nose (Fig. 2C). Both the duration of the quiescent period and
rate of growth after axotomy in the L1 were comparable with that
after axotomy in the L4 (Fig. 2E). ALM neurons cut in the L2
stage also regrew to the nerve ring, although slightly less
frequently (3/11). Overall, ALM axons cut in the L1/L2 stages
were less likely to reverse in the anteroposterior (AP) axis or
extend posterior processes than when they were cut in L4 or
young adult (3/22 L1/L2 vs. 11/18 L4/adult, P ⬍ 0.01 by Fisher
exact test). Touch neurons cut in early larval stages were less
likely to extend additional branches than those cut in later stages
(3/16 touch neuron axons extended side branches 24 h after L1
axotomy, versus 19/37 in L4 stage; P ⬍ 0.05).
After its primary outgrowth in the embryo, the ALM axon
grows proportionally during larval development (22). The ALM
axon, measured from cell body to synaptic branch, grows from
74.2 ⫾ 4 ␮m in the L1 to 217 ⫾ 7 ␮m in L4, and to 303 ⫾ 13 ␮m
in 1-day old adults (n ⫽ 7). The reduced ability of later larval
axons to reach their targets could in part reflect decreased
proportional growth as opposed to growth-cone dependent
15134 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0707001104
54 h
*
C
33
20
b
22
*
*
30
20
11
00
ALMR
DV deviation (µm)
*
df
DV
df
b
9h
growth ratio
synaptic
branches
X
40
wt
wt
11
9
dx14
e2027
e118
efn-1
dx14
vab-1
e2027
efn-1
vab-1(0) (e118) (ju1)
20
E
15
*
10
10
**
anterior posterior
MYR::VAB-1
13
8
MYR::VAB-1(G921E)
5
11
0
0
WT L4
wt
e2027 L4
e118 L4
vab-1(0) vab-1(kd)
100
µm / 48 h
zdIs5
100
Fig. 3. Eph signaling influences guidance of regrowing touch neurons. (A) PLM
processes cut in L4 stage proximal to the branch point by using MHz laser, marker
zdIs5. Note prominent growth cones at 9 –25 h and posterior turning between 25
and 56 h. (B) PLM axons turn less in vab-1(e2027) background; b, PLM branch.
(Scale bar: 10 ␮m.) (C) Growth ratio (total distance grown by axon divided by net
distance from cut site to tip of axon) in wild type, vab-1 null and kinase-dead
(e118) mutants, and efn-1(ju1). The total regrowth of PLMs at 24 h was not
significantly different (wt, 100 ⫾ 18 ␮m; vab-1, 64 ⫾ 8 ␮m; P ⫽ 0.09 by t test). (D)
maximum dorsoventral distance (DV, dashed line in 56 h panel) of regrown
processes from distal fragment, in wild type, vab-1(e2027), vab-1(e118) animals
cut as L4s and scored 48 h later. (E) Regrowth of anterior and posterior processes
after anterior axotomy in MYR::VAB-1 (quIs5) and MYR::VAB-1(G912E) (quIs4) L4
animals, and WT controls; all strains contain the zdIs5 marker. Anterior regrowth
is significantly reduced only in quIs5 expressing animals. *, P ⬍ 0.05; **, P ⬍ 0.01.
outgrowth. However, the total increase in process length after
axotomy was not significantly different in L4 versus L1 animals
(159 ⫾ 23.8 ␮m in L4 vs. 178 ⫾ 16.4 ␮m in L1, between 0 and
48 h; n ⫽ 7). Only adults showed a decreased growth rate relative
to L1s (Fig. 2E). Finally, after axotomy in the L1 and L2 stages
the distal fragments became thin, beaded and often invisible
within 24 h (Fig. 2C). By contrast, after axotomy in L4s or young
adults, the distal fragment became fainter and had a beaded
morphology, yet remained visible for several days, suggesting
that the rate of removal or degeneration of the distal fragment
also decreases with larval stage. We conclude that the primary
reason regrowing mechanosensory neurons fail to reach their
targets in later larval stages is their aberrant guidance, although
decreases in axon growth and the persistence of distal fragments
may also play a role.
Eph Receptor Signaling Affects Guidance of Regrowing PLM Processes.
The aberrant guidance of touch neurons in later larval stages
could be due to loss of their normal guidance cues, or it might
reflect inappropriate or elevated responsiveness to other cues.
Ephrins are conserved axon guidance cues in vertebrates and in
C. elegans (23), and ephrin signaling influences regenerative
axon growth in vertebrates (24). In C. elegans mutants lacking the
VAB-1 Eph receptor the PLM axon sometimes overshoots (25).
We therefore tested whether ephrin signaling might contribute
to guidance errors in regrowth of the touch neurons. The rate of
PLM regrowth was not significantly different in vab-1 null
mutants compared with wild type (mean rates 3.1 ⫾ 0.5 ␮m/h in
zdIs5 (n ⫽ 12), 2.1 ⫾ 0.4 ␮m/h in vab-1 zdIs5 (n ⫽ 18); P ⫽ 0.15).
However PLM regrowth in vab-1 mutants was straighter than in
the wild type: in vab-1 mutants axotomized PLM axons rarely
reversed direction in the AP axis and remained closer to their
original trajectory (Fig. 3B). To quantify these effects on guidWu et al.
The Synaptic Branch of Touch Neurons Marks a Transition in Regenerative Capacity. The synaptic output of mechanosensory neurons
is confined to their collateral branches to the nerve ring (ALM)
or to the ventral cord (PLM). Studies on C. elegans synapsedefective mutants have suggested that formation of stable synaptic branches may repress axon growth (27). We therefore
tested how the synaptic branch influences regrowth after axotomy. We first cut PLM axons immediately proximal or distal to
their synaptic branch in the L4 stage. When we cut PLM 10 ␮m
proximal to the branch, it consistently regrew as described above
(Figs. 3A and 4E). By contrast, when we cut the PLM axon 10 ␮m
distal to its branch, almost no regrowth occurred up to 48 h post
cutting (Fig. 4A, 3/25, zdIs5; 0/11, muIs32). The PLM synaptic
branch also did not regrow when cut (Fig. 4B; 0/8 zdIs5, 0/7
muIs32); instead, the stump of the branch retracted to the axon
such that the branch could not be found after 24 h. Cutting the
axon distal to the branch occasionally resulted in a small amount
of growth of the branch (Fig. 4 A and E); however, cutting the
branch alone had no significant effect on growth of the distal
axon (data not shown). The inhibitory effect of the synaptic
branch is not unique to PLM, as we observed a similar effect of
the ALM synaptic branch (SI Fig. 8 B–D).
In the course of these experiments we observed that the
position of the PLM branch varied depending on the transgenic
background used. In muIs32 animals the PLM branch is in its
correct position, just posterior to the vulva, whereas in zdIs5 the
PLM branch is more posterior. Axons cut proximal to the branch
in muIs32 animals regrew, even though they were further from
the cell body than distal axotomy sites in zdIs5.
Wu et al.
Fig. 4. The touch neuron synaptic branch inhibits regrowth of the distal
axon. All animals were cut in mid L4 stage, zdIs5 marker. (A) The PLM distal
process does not regrow after cutting; in some animals, the synaptic branch
grows in response (*). (B) Retraction of PLM synaptic branch stump after
cutting (arrow). (C) The distal process regrows when the branch is also cut. (D)
Distal axon regrowth stimulated when the branch is cut 12 h after the distal
axon. Anterior is to the left and dorsal up in all panels. (Scale bars: A–C, 5 ␮m;
D, 10 ␮m.) (E) Process regrowth in 24 h after kHz or MHz axotomy of proximal
(P), distal (D) axon, or distal ⫹ branch (D⫹B), or branch point (BP); also plotted
is branch growth after cutting distal axon [D(B)]; n ⫽ 6 –15 for each group;
comparisons use t test. ***, P ⬍ 0.001; ns, not significant. Axon growth after
the delayed branch cut (D) is measured between 24 and 48 h after the first cut.
PLM axons regrew less when cut far from the cell body
compared with cutting closer; we found a similar effect of
absolute distance in ALM (SI Fig. 8E). Thus, although axons cut
further from the cell body regrow less, this effect is independent
of the influence of the synaptic branch point.
The synaptic branch of ALM/PLM marks a transition between
the proximal axon, which can regrow after injury, and the distal
axon, which cannot. Because cutting the branch itself caused it
to retract and disappear, we tested whether cutting both the
distal axon and the branch could reverse the inability of the distal
axon to regrow. Strikingly, when the branch was also cut, the
distal axon of PLM regrew to an extent similar to that seen after
cutting the proximal axon (Fig. 4 C and E). Ablation of the
branch point itself also resulted in regrowth from the proximal
stump (BP, Fig. 4E). These findings suggest that axotomy results
in an intrinsic change to the cell. To test whether such a change
persists in time we cut the PLM branch 10–12 h after cutting the
distal axon, and found that this also strongly stimulated distal
regeneration, equivalent to that after simultaneous cutting (Fig.
4 D and E). We infer that cutting the distal axon results in a
long-lasting change to the cell, and that regrowth from the distal
stump is actively blocked by the presence of the synaptic branch.
Discussion
We show that multiple C. elegans neuron types can regenerate
after laser induced damage. Motor and sensory neurons also
undergo regenerative growth in C. elegans ␤-spectrin mutants, in
which axons undergo cycles of breakage and regeneration because of axon fragility (28). Regeneration of injured neurons is
common in other invertebrates (29), and our findings reinforce
the view that C. elegans neurons have a high capacity for
regrowth after injury. As in other animals, the propensity of a C.
elegans neuron to regenerate is affected by a complex set of
environmental and intrinsic factors.
Axons cut using laser axotomy undergo a stereotyped sequence of events. At the time of cutting a break of less than a
PNAS 兩 September 18, 2007 兩 vol. 104 兩 no. 38 兩 15135
NEUROSICENCE
ance we calculated the ratio of the total distance extended by the
axon divided by the net distance, measured as a straight line from
the site of axotomy to the tip of the axon. In the wild type the
regrowing axon extended on average 2.9 times its net distance
whereas vab-1 axons extended 1.1 times their net length (P ⫽
0.04, unpaired t test; Fig. 3C). These results suggest impaired
guidance of PLM regrowth in adults is due to Eph signaling. Eph
receptors are bidirectional signaling molecules that can stimulate
‘‘forward’’ kinase-dependent signaling into the Eph receptorexpressing cell, or reverse signaling into ephrin-expressing cells
(26). To test whether the misguidance of regrowing axons
required forward or reverse Eph signaling we cut PLM in
animals lacking VAB-1 kinase activity [vab-1(e118)] and observed wild-type like deviation (Fig. 3 C and D). This result
suggests that the abnormal guidance of PLMs may involve
kinase-independent functions of VAB-1 Eph receptor. Axotomy
of animals lacking the EFN-1 ephrin also resulted in wild
type-like misguidance (Fig. 3C). As ephrins have redundant roles
in developmental PLM outgrowth (25), they may also be redundant in regrowth.
Expression of a myristoylated VAB-1 intracellular domain
(MYR::VAB-1) in touch neurons causes premature termination of
PLM axon outgrowth because of constitutive activation of the
VAB-1 kinase-dependent ‘‘forward signaling’’ pathway (25). We
tested whether VAB-1 forward signaling might also influence PLM
regrowth, as opposed to guidance. Axotomized PLM axons expressing MYR::VAB-1 (quIs5) displayed significantly less anterior
regrowth than the wild type (45.52 ⫾ 14.5 ␮m quIs5 vs. 125.0 ⫾ 13.4
␮m WT outgrowth in 48 h, P ⬍ 0.01 by ANOVA) or animals
expressing a control kinase-dead MYR::VAB-1(G912E) (quIs4;
Fig. 3E). Instead, MYR::VAB-1 expressing PLM neurons frequently extended their uninjured posterior axons (Fig. 3E). We
infer that activation of VAB-1 forward signaling does not globally
inhibit regrowth but may elevate sensitivity of the anterior axon to
repellent cues, triggering growth of the uninjured axon. Taken
together, VAB-1 signaling seems to play two distinct roles in PLM
regrowth: a kinase-independent role in guidance, and a kinasedependent role that represses anterior growth.
micrometer in length is made; these breaks expand over the
course of the next few hours. These short-range retractions
appear distinct from the acute degeneration of axons observed
by using live imaging in mice, where cut ends die back hundreds
of microns within 30 min of injury (30). Fusion of the cut ends
was more often seen after the more precise cutting by using
amplified lasers (21), but less so after MHz axotomy, which
creates an area of damage that likely physically blocks the
regrowing process. Nevertheless in several animals we observed
the regrowing process grow around the damaged area and fuse
with the distal fragment, suggesting regrowing axons can recognize their distal fragments and will tend to fuse with them if they
form contacts.
After axotomy an axon typically displays a quiescent period of
between 6 and 24 h, after which a new growth cone can be seen
at the proximal stump. Motility of the regenerated growth cones
was highest over the next 36 h and then declined. In our
experiments axons regrew at up to 10 ␮m/h; if regrowing growth
cones move discontinuously then their actual maximum rate may
be higher. As VD growth cones can travel up to 60 ␮m/h in intact
animals (31) these results suggest that regenerating C. elegans
axons grow more slowly than in normal development.
Developmental Changes in Regrowth. We found two main differences in the regrowth of early larval versus late larval or young
adult axons. The amount of regrowth of touch neurons declined
slightly from L1 to L4 stages; this seemed to reflect a longer
quiescent period before regrowth commenced, as L4 stage axons
eventually could regrow at the same rate as L1 axons. Only in
young adults did the maximum rate of extension decline. Developmental changes in axon extension rates have been observed
in several organisms (32–34) and may contribute to declines in
ability to regenerate. Our findings suggest that such developmental declines can be analyzed even in the rapidly developing
C. elegans nervous system.
Second, guidance errors in regrowth increased with age.
Several factors could account for the decrease in accuracy.
Regrowing axons have further to go in later stages, and thus have
more potential room for error. If guidance cues for the touch
neurons are distributed in gradients these may be less effective
if spread out over the larger scale of the L4 animal. Touch
neurons also undergo ensheathing by surrounding epidermal
cells that could block extracellular signals, leading to inaccurate
regrowth (35). Finally, aberrant sensitivity to cues such as the
ephrins may contribute to guidance errors. Dorsal growth of D
commissures in embryos and L1 animals depends on repulsion
from a ventral source of UNC-6/netrin (36). As UNC-6 also
directs later dorsal guidance events such as the migration of
gonadal distal tip cells in the L3 stage (37), it is likely that enough
UNC-6 gradient remains in the L4 stage to accurately guide D
neurons.
Eph Signaling and Regeneration. In wild type late larvae and adults,
regenerating touch neuron axons deviated far from their original
trajectory and often reversed direction. vab-1 mutant axons
deviated significantly less and showed significantly fewer reversals. Misguidance of regenerating PLMs in the wild type could
reflect increased sensitivity of the regenerating axons to repellent cues in the anterior, consistent with a repulsive role for Eph
signaling in PLM termination. As regenerating PLMs typically
reverse before their normal termination point, such repellent
cues may be broadly distributed. We speculate that anteriorly
localized ephrins regulate both the direction of growth and the
termination position of the PLM neurons. C. elegans ephrins are
widely distributed in neuronal and epidermal cells (38, 39), and
further work will be required to determine their cellular focus in
touch neuron regrowth. Ephrin signaling both promotes and
inhibits regeneration in vertebrates. Regeneration of a topo15136 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0707001104
graphic retinotectal map in amphibians is likely dependent on
expression of ephrins and their receptors (40), and up-regulation
of EphB3 after injury contributes to the regrowth of retinal
ganglion cells (41). By contrast, ephrin-B3 is an inhibitory
component of CNS myelin (24), and elimination of its receptor
EphA4 enhances regeneration of the corticospinal tract (42).
Role of Branches. We find a sharp transition in regenerative ability
at the touch neuron synaptic branch, independent of distance
from the cell body. What is it about the synaptic branch that
exerts such a strong effect on regrowth? Because cutting the
branch at any position causes the stump to disappear, it is unclear
whether the branch junction, the branch itself, or synapses made
by the branch, account for this effect. In animals lacking the
RPM-1 ubiquitin ligase, PLM synaptic branches extend normally
but later retract; concomitantly the axon itself grows into the
ventral nerve cord (27). The rpm-1 phenotype suggests the PLM
branch or its synapses inhibit axon growth. However, axotomy of
the branch alone does not stimulate growth of the distal axon.
Retraction of the branch stump may eliminate a ‘‘damage’’ signal
that would otherwise lead to regrowth. Alternatively, inhibition
of axon growth by synaptic signaling may only be effective in a
critical period of synaptogenesis.
We speculate that a sorting area at the PLM branch point
routes presynaptic components to the branch and not further
along the axon. After the distal axon is severed, growth is
stimulated, presumably requiring increased delivery of membrane from the soma. When the branch is present such components are mostly sorted to the branch. In contrast, when the
branch is absent the sorting area disappears and the distal axon
is now able to grow at the site of axotomy. Branch points of
dendritic arbors contain Golgi outposts that may play a role in
dendrite growth (43, 44). A Golgi outpost like structure at the
PLM branch point could participate in sorting to the presynaptic
branch. If this structure requires the synaptic branch itself for
stabilization, loss of the branch by axotomy or lack of RPM-1
may lead to loss of sorting and of the distinction between distal
and proximal in the axon. Tests of this model will require
identification of molecules acting at the branch point itself. The
effects of cutting the branch may also be analogous to ‘‘conditioning lesion’’ paradigms (12), in which axotomy of a peripheral
branch stimulates regrowth of a central axon. Further analysis
should reveal whether additional similarities exist at the level of
molecular mechanism.
Materials and Methods
Genetics. C. elegans strains were maintained on nematode growth
medium (NGM) agar plates using standard procedures at 20°C–
23°C. We used the following mutations: vab-1(e2027, dx14, e118)
(45), efn-1(ju1) (39). To visualize D type motor neurons, we used
Punc-25-GFP juIs76 (46); to mark DDs, we used Pflp-13-GFP
transgenes juIs145 (J. Meir and Y.J., unpublished results) or
ynIs37 (47). We used two transgenes to mark the touch neurons:
zdIs5 (Pmec-4-GFP) and muIs32 (Pmec-7-GFP). zdIs5 drives
GFP expression in the six touch neurons (48); muIs32 is also
weakly expressed in several other cell types. To mark phasmids
PHA and PHB, we used Psrb-6-GFP gmIs12 (49) and to mark the
AWB neurons we used Pstr-1-GFP kyIs104 (50).
Femtosecond Lasers. In most experiments described here, we used
a KMLabs MTS Ti-Sapphire oscillator (Kapteyn-Murnane Laboratories, Boulder, CO) pumped by a Verdi V5 (Coherent, Santa
Clara, CA). The measured pulse energy of this oscillator was 3.4
nJ at a repetition rate of 94 MHz and wavelength of 790–800 nm.
We used a home-built autocorrelator to measure a pulse duration of 150 fs (FWHM). In more recent experiments, we used a
KMLabs Cascade laser that can be operated in mode-locked
continuous wave (80 MHz) or cavity-dumped (1–100 kHz)
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PNAS 兩 September 18, 2007 兩 vol. 104 兩 no. 38 兩 15137
NEUROSICENCE
modes (SI Table 1). In some configurations, we used a spatial
filter to clean and expand the beam. We attenuated the pulse
energy using neutral density filters and controlled pulse delivery
with an electro-mechanical shutter (Uniblitz VS14 with
VMM-T1 controller; Vincent Associates, Rochester, NY). The
laser beam enters the side port of a Zeiss Axiovert 200 (Carl
Zeiss, Jena, Germany) equipped with a dual camera attachment
and reflects off a mirror and through a Planapo ⫻100x/N.A. 1.4
objective to the specimen. GFP-labeled axons were visualized by
using epif luorescence and a Hamamatsu Orca camera
(Hamamatsu Photonics, Hamamatsu City, Japan) controlled by
Improvision Volocity software (Improvision, Lexington, MA).
Custom filters (Chroma Technology, Rockingham, VT) were
used to ensure transmission of the laser beam. To anesthetize
animals for surgery and imaging, we used 0.1–1% 1-phenoxy-2propanol (TCI America, Portland, OR) in M9 buffer and in the
agar pad.
After axotomy, we occasionally observed faint GFP expression
in nearby cell bodies and processes, beginning several hours after
cutting. Such ectopic expression may be due to laser-induced
membrane fusion and GFP mixing between the axotomized cells
and adjacent processes. Membrane fusion induced by UV laser
irradiation has been observed in C. elegans embryos and oocytes
(51, 52). We scored regrowth of a cut axon in such cases only
when it could be unambiguously distinguished from ectopic GFP
expression in a neighboring process.